Use of mononuclear phagocytes in in vivo imaging of hypoxic/ischaemic tissue

ABSTRACT

The invention relates to the imaging, preferably of hypoxic or ischaemic sites using mononuclear phagocytes. Specifically, the migratory behavior of the mononuclear phagocytes is exploited with a view to targeting imaging agents to sites that mononuclear phagocytes penetrate.

[0001] The invention relates to a method of delivering imaging agents;means therefor including components thereof which have particular, butnot exclusive, application in the development of therapies for cancer orcoronary heart disease.

[0002] Macrophages often comprise 20-60% of the tumour cell mass inbreast carcinomas and form intimate contacts with malignant cells. Thishas long been thought to represent part of the host's defence mechanismsagainst the tumour; however, their function at such sites in the bodyremains an enigma at present as macrophages isolated from human ormurine turnouts exhibit reduced tumouricidal, phagocytic andantigen-presenting activities compared to those from normal tissues (1).

[0003] Monocytes are produced in the bloodstream and extravasate (i.e.exit) into surrounding tissues including such diseased tissues asmalignant tumours and atherosclerotic plaques, where they differentiateinto macrophages and perform immune, secretory, phagocytic and otherfunctions. Monocytes and macrophages are collectively termed mononuclearphagocytes. As tissue macrophages have a lifespan of 60 to 90 days andthe number of macrophages in tumours remains constant, it is believedthat there is a constant attachment of monocytes to the tumourendothelium and influx of monocytes into the tumour cell mass.

[0004] Hypoxia, that is, very low levels of oxygen, exist only in someforms of diseased tissue (e.g. malignant tumours, ischaemic hearttissue, retinal tissue etc.) (2). Hypoxia and/or hypoglycaemia isthought to occur in (growing tumours when the increasing metabolicdemands of the rapidly expanding tumour cell population outstrip thesupply of oxygen/glucose etc., made available to them by simplediffusion across the tumour mass from vessels in surrounding normaltissues.

[0005] Recent and surprising data indicate that once monocytes enter atumour from the bloodstream, they rapidly differentiate into macrophagesand preferentially congregate in hypoxic (i.e. poorly vascularised andnecrotic) sites deep within a tumour mass remote from blood vessels.Refer to FIG. 1, which represents a bar chart of the Distribution ofMacrophages in Relation to Blood Vessels. Moreover, breast tumours, withmore hypoxic/necrotic areas, are more heavily infiltrated withmacrophages, which preferentially locate to, or around, the necroticsites (refer to FIG. 2, which represents a bar chart of the Associationof Macrophage Index with Necrosis in Breast Carcinomas). Experimentalhypoxia has been shown to induce the production of angiogenic factors bymacrophages in vitro (3). Taken together these data could underpin ourrecent finding, that increased numbers of macrophages in breast tumoursequate with increased fatalities in breast cancer (4).

[0006] We have also shown recently that human macrophages accumulatespecifically in cell layers immediately adjacent to the central areas ofnecrosis in three-dimensional cultures of human cancer cells. Manyprevious studies have shown that this viable rim of tumour cells aroundthe necrotic core of such spheroids are severely hypoxic relative to theouter layers of tumour cells in these cultures (5). That macrophagescongregate in hypoxic diseased tissues other than malignant tissue hasbeen shown for coronary heart disease (6). as well as suchcerebrovascular disorders of the central nervous system as strokes andcerebral malaria (7).

[0007] The observation that tumour and other forms of ischaemic tissueare regions of poor oxygenation has lead to the development of a numberof techniques to assess oxygen tension in these tissues. Invasivesurgical procedures include the insertion of polarographicmicro-electrodes into tumour tissue to measure directly the levels ofoxygen in a given tissue (2). Non invasive techniques have also beenadopted which involve the use of radiopharmaceuticals (eg F-18Fluoromisonidazole) which bind to hypoxic cells (8). The concentrationof the agents are then detected and quantified by methods such as wholebody positron emission tomography (PET imaging) (9). A major problemwith this imaging technique is that radiopharmaceuticals tend to beneurotoxic due to their lipid solubility. Clearly this problem would beovercome if it were possible to bind these products to a delivery means.A further major problem with this imaging technique is the poor level ofresolution achieved by radiopharmaceuticals due to a relatively highbackground detection in tissues that do not have appreciable levels ofhypoxia. An improvement of the level of detection in hypoxia sites canbe achieved if sufficient time is allowed for the clearance of theradiopharmaceutical from non target tissues. However this can takeseveral hours to achieve and is therefore not a desirable situation.

[0008] The current state of the art describes a number of means toenhance the localisation of imaging agents to hypoxic and/or ischaemicsites. In broad terms current techniques involve the encapsulation ofimaging pharmaceuticals within microvesicles. Alternatively the imagingagents can be directly modified to enable either the localisation of theagent to the desired tissue or enhance their detection when the agentaccumulates in the target tissue.

[0009] Typically microspheres encapsulating an imaging agent areliposomes composed of either pure phospholipid or a mixture ofphospholipid and phosphoglyceride. They are advantageous due to the easewith which the microspheres can be produced containing the imagingpharmaceuticals. By altering conditions during manufacture microspherescan be produced that have diameters of less than 200 nM which enablesthem to be intravenously injected and able to pass through the pulmonarycapillary bed. Furthermore the biochemical nature of the liposomeconfers permeability across blood vessel membranes to access the tumoursite or region of ischaemia. Liposomes of this type show highechogenicity both in vitro and in vivo which would be a necessaryrequirement using techniques of magnetic resonance imaging (11),fluoroscopy and computerised tomography (10).

[0010] However this technology does suffer a major disadvantage in thatthe liposomes lack an intrinsic affinity for the targeted tissue andrelies on a local intravenous injection of the liposome composition inthe vicinity of the diseased tissue.

[0011] What patients require is a rapid and accurate diagnosis of theircondition so that an effective treatment regime can be established asquickly and accurately as possible. The development of an effectivemeans of targeting imaging means to hypoxic/ischaemic sites wouldobviously benefit both clinicians and patients in the diagnosis andtreatment of diseases such as cancer and coronary heart disease.

[0012] An alternative strategy is to chemically modify an agent that hasa natural affinity for tumour/ischaemic tissue to enable the detectionof the agent at the targeted tissue.

[0013] Monosaccharide derivatives have been used as imaging agents(patent application no. WO.9634872-A). Glucose levels have been shown tobe an important indicator in diagnosis of Alzheimer's disease, Parkinsondementia, epilepsy, diabetes and myocardial ischaemia. The elevatedlevels of glucose consumption in tumour ischaemic tissue has beenexploited by using iodinated glucose to identify these regions. Althoughmodified monosaccharides have excellent in vivo stability they have ageneral biodistribution in the body and problems with optimising thesignal to noise ratio during treatment can arise.

[0014] The labelling of peptides with technetium-99m and there detectionvia scintigraphic imaging has been used in the diagnosis of tumours(patent application no. WO.9310747-A). Peptides are typically composedof 4-100 amino acid residues. The technetium-99m labelled peptides havebeen successful used to diagnose kidney disorders by scintigraphicimaging. However although imaging peptides have excellent in vivostability they lack an intrinsic targeting property making resolutionsomewhat problematic.

[0015] More recently the use of a radioactive copper complex ofdithiosemicarbazone has been employed to image regions of hypoxia and/ormitochondrial dysfunction (EP-726077-A). The composition is advantageousdue to improved permeability and retention in target cells but with ashort residence in non-targeted cells. The imaging potential of thiscomposition is improved due to the fact that it is only reduced intissue containing an excess of electrons (eg tissues that containdysfunctional mitochondria). However although copper containingdithiocarbazone shows retention in hypoxic tissue there are stillsignificant levels of the composition in non-targeted normoxic tissuethus reducing detection resolution.

[0016] Finally, the use of monoclonal antibodies to target radioactiveand non radioactive imaging agents has been exploited. The expression ofspecific membrane proteins has lead to the production of monoclonalantibodies to these membrane proteins to enable the targeting of imagingagents to tumour tissue. However this advantage is offset by the pooraccess of the tagged antibodies to the tumour tissue.

[0017] In summary the compositions that have been described either lackan intrinsic means of targeting the imaging agent to a diseased tissueor have poor access to the sites of hypoxia/ischaemia. This leads to apoor signal to noise ratio resulting in reduced imaging resolution.

[0018] It is therefore an object of this invention to identify a meansto target imaging agents to regions of hypoxia/ischaemia which exploitsthe fact that mononuclear phagocytes have an affinity for regions ofhypoxia/ischaemia.

[0019] The invention in its broadest aspect, comprises the use ofmononuclear phagocytes to deliver conventional imaging agents to tissuesand especially hypoxic/ischaemic sites.

[0020] According to a first aspect of the invention there is thereforeprovided an imaging means comprising an imaging agent attached to anagent that binds to a cell surface element of a mononuclear phagocyte.

[0021] The invention comprises the conjugation of imaging agents to thesurface of macrophages via macrophage specific cell surfaceproteins/receptors (eg CD68, CD87, CSF-1). The imaging agent could beattached via a monoclonal antibody, ideally humanised, to one or more ofthese cell surface proteins or to a ligand specific for a particularmacrophage cell surface marker. The macrophages could be modified eitherin vivo or ex vivo and reintroduced into the patient to allow macrophagemigration into the hypoxic/ischaemic tissue. It may be advantageous, butnot always necessary, to use an imaging agent that becomes more readilydetected due to the conditions of hypoxia or ischaemia.

[0022] In a preferred embodiment of the invention said imaging agent isof a conventional nature such as, without limitation, an imagingpharmaceutical such as a radiopharmaceutical or a technetium-99mpeptide.

[0023] It is therefore a further object to provide a novel imaging meansthe detection of which is enhanced by hypoxic/ischaemic conditions.

[0024] According to a farther aspect of the invention there is providedan imaging means comprising a hypoxia enhanced imaging agent and anagent that binds to a cell surface element of a mononuclear phagocyte.

[0025] It will therefore be apparent that the hypoxia enhancing imagingagent will be affected by hypoxic conditions and typically affected soas to lead to enhanced detection in such conditions. A typical exampleof this sort of agent is described in WO 9634872-A. Moreover, saidbinding agent, which is typically coupled to said imaging agent,attaches the composition to mononuclear phagocytes and so targets theimaging agents, to sites typically infiltrated by mononuclearphagocytes. Thus in the instance where the said mononuclear phagocytespenetrate hypoxic sites said composition is suitably delivered to suchsites and the imaging agent shows enhanced detection.

[0026] The invention is elegant in so far as the body's own mechanismsare exploited for the specific delivery of the imaging agent to regionsof hypoxia or ischaemia.

[0027] Given the above nature of the invention agents suitable for usein manufacturing the composition will be known to those skilled in theart and therefore the following preferred embodiments are not intendedto be exhaustive but rather illustrative.

[0028] It may be preferable to conjugate said imaging agent to a carriermolecule that promotes the internalisation of the imaging compositioninto mononuclear phagocytes. Internalisation signals include, but arenot limited to, plasminogen activation inhibitors (PAI-1 PAI-2) orprotease nexin (PN), which bind to, and cause the internalisation ofCD87 (the receptor for Urokinase Plasminogen Activator) into monocytesand macrophages.

[0029] In a preferred embodiment of the invention said binding agent isadapted to bind to any one or more cell surface mononuclear phagocytemolecules such as antigens or receptors.

[0030] Further, said binding agent may comprise an antibody to any oneor more of said molecules such as antigens or receptors, or an effectivefragment of said antibody. Alternatively still said binding agent maycomprise a suitable ligand either synthetically manufactured ornaturally occurring. For example, chemicals such as benzodiazepines andPK1195 bind to a specifc receptor on the surface of macrophages (Zavalaand Lenfant (1987) Annals N Y Acad Sci 496, 240-249).

[0031] A brief list of those cell surface molecules that may be targetedby said binding agent is as follows; CD87; the receptor for human ColonyStimulating Factor (CSF-1); CD11b; CR3; the scavenger receptor; all orpart of the receptor for the various forms of human monocytechemoattractant protein (MCP-1,2, etc); CD14; mannose or mannose -6-phosphate surface receptors.

[0032] According to yet a further aspect of the invention there isprovided a delivery system for targeting imaging compositions to hypoxicor ischaemic sites comprising an imaging agent and, optionally, an agentfor controlling the functional effectiveness thereof, and coupledthereto, a binding agent for a cell surface molecule of a mononuclearphagocyte.

[0033] According to yet a further aspect of the invention there isprovided a method for targeting imaging agents to hypoxic or ischaemicsites comprising;

[0034] (i) coupling at least one of said agents to a binding agent of acell surface molecule expressed by a mononuclear phagocyte;

[0035] (ii) exposing said coupled agents to the mononuclear phagocytes;and

[0036] (iii) allowing the said phagocytes to migrate under conditionsthat support migration in vivo.

[0037] According to yet a further aspect to the invention there isprovided a method for imaging hypoxic or ischaemic sites comprisingadministering to an individual to be treated the imaging means of theinvention.

[0038] According to yet a further aspect of the invention there isprovided mononuclear phagocytes having coupled thereto, or internalisedtherein, an imaging agent and an agent that is adapted to bind to amononuclear phagocyte ligand which is typically found on the cellsurface of the said mononuclear phagocyte.

[0039] In essence the invention describes the use of mononuclearphagocytes to deliver imaging agents to regions of hypoxia/ischaemia toincrease the resolution of detection of said imaging agent eitherthrough localisation or by use of hypoxia enhanced imaging agent.

[0040] An embodiment of the invention will now be described by way ofexample only with reference to the following wherein:

[0041]FIG. 1 shows the distribution of macrophages in relation to bloodvessels

[0042]FIG. 2 shows the association of macrophage index with necrosis inbreast carcinomas.

[0043]FIG. 3 shows macrophage infiltration into hypoxic areas in tumourspheroids (i.e. an in vitro model of tumour hypoxia imaging);

[0044] (i) shows the oxygen profile across a tumour cell spheriod. Allbut the cells in the outer 100 um of these 3-D cultures are hypoxic(i.e. experiencing oxygen levels of 0-15 pO₂ mmHg; a level equivalent tothat present in hypoxic/necrotic sites in human tumours). This hypoxiais produced by the inability of oxygen to diffuse into the central areasof spheroid. The glucouse profile of the spheriod is similar to thatseen for oxygen.

[0045] (ii) shows two different tumour spheriods (made of the breastcancer cell line, MCF-7) following co-culture for 24 h with themonocytic cell line, U937. The U937 cells (darkly stained cells labelledwith a monoclonal antibody to the pan-macrophage marker, CD68)accumulate in the hypoxic rim of viable, but hypoxic tumour cells aroundthe central areas of necrosis (“N”).

[0046] (iii) shows the infiltration into tumour spheroids of U937 cellspreloaded with fluorescent dye. The top panel is a light micrographshowing the opaque

[0047] (iv) central area of necrosis (“N”) which forms in thesespheriods as a consequence of nutrient (e.g. oxygen, glucose etc)deprivation. The bottom panel is the same spheriod using a fluorescentmicroscope to show the presence within the spheriod of the fluorescent(i.e. light coloured cells) U937 cells. The latter take up a similarposition to that seen in (ii), i.e. they congregate in a collar ofhypoxic tumour cells around the central areas of necrosis.

Materials and Method

[0048] Infiltration of multi-cellular human tumour spheriods with humanmacrophages (U937 cells or monocytes).

[0049] Tumour spheriods were established in culture using the MC7 cellline (ATCC) using the following procedure.

[0050] A. Establishment of Spheroid Cultures

[0051] 1. Uniformly sized spheriods were grown in standard 96-welltissue culture plates.

[0052] 2. A 1.5% solution of agarose was prepared in media andautoclaved (the medial should not contain any supplements or foetal calfserum as this causes the formation of bubbles and the cells will platedown and not form spheriods).

[0053] 3. 100 μl of the agarose was aliquoted into each well and allowedto cool. Plates were then warmed to 37° C. before use.

[0054] 4. Monolayers of tumour cell lines were stripped in theexponential growth phase, resuspended and counted using, ahaemocytometer. The cells were then diluted to the appropriate number ofcells for spheroid initiation. For T47D and HT29 this was 1000 cells perwell and for MCF-7 it was 2000 cells per well. Each well was filed with200 μl of the cell suspension. The final concentration of the cellsuspension for T47D was 5000 cells per ml. (NB spheriods were grown inthe media used normally for each of the cell lines (eg, T47D are grownin DMEM supplemented worth antiobiotics and fungicides).

[0055] 5. Following, initiation, the spheroids were incubated at 37° C.in a CO₂ incubator and left undisturbed for 5 days to allow aggregationto occur.

[0056] 6. Spheroids were fed fresh medium three times per week.

[0057] B. Co-culture of spheroids with macrophages

[0058] 1. Moncytic cell lines eg U937 cells), peripheral blood moncytesor moncyte-derived macrophages were introduced into the spheroid oncethe spheroids have formed necrotic centres. This stage depends upon thecell line used. For MCF-7 and T47D it was after 2-3 weeks of culture,when a dark area can be seen in the centre of the spheroid.

[0059] 2. This was done by removing 100 μl of media from the wells andreplacing it with a suspension of macrophages (50,000 per well). Forcell lines, the change in media was not a problem but for PMBC the mediashould be serially changed for both the spheroid and the macrophageuntil they are in the same media. These cells infiltrated the spheroidsin the first hour of co-culture and continued to do so for up to 48 hrs.After this, the spheroids were removed from the wells using a glass ofPasteur pipette, placed in a test tube and rinsed in PBS to remove anyloose macrophages or cell debris.

[0060] 3. The spheroids were then allowed to settle to the bottom of thetube, the PBS removed and the spheroids processed for paraffin embedding(or frozen in OCT).

[0061] The results of an experiment showing infiltration of MCF-7spheroids with U937 cells is shown in FIG. 3. Each spheroid displays thetypical central area of necrosis (‘N’) surrounded by a collar of hypoxictumour cells (approx 5-10 cells in thickness) and then several outerlayers of cells that are relatively normoxic. U937 cells,immuno-labelled (dark staining) for the pan macrophage marker CD68, canbe seem accumulating in the hypoxic tumour cell layers around thecentral necrosis.

[0062] Demonstration of infiltration into spheroids of monocytic cellslabelled with a fluorescent dye.

[0063] U937 cells were incubated with 90 μl of4-(4-(didecylamino)styryl-N-methyl pyridium iodide (2 mg/ml in absethanol) for 45 minutes then washed to remove the excess dye. Multicellspheroids (5-600 μm in diameter) were placed in a bacterial petri dish(agarose would flouresce and they would adhere to tissue cultureplastic) and incubated with 75000 dye-loaded U937 cells per spheroid.The final volume was 15 mls. After 4 days of co-culture, spheroids werewashed to remove unattached macrophages and photographed using afluorescence microscope.

[0064] The result is shown in FIG. 3(iii). Panel A in FIG. 3(iii) showsa section through a spheroid photographed under white light. The centralnecrotic core (N) is visible as a dark area. Panel B, photographed underfluorescence optics, shows accummulation of macrophages labelled withfluorescent dye, in the hypoxic region around the necrotic core.

[0065] C. Paraffin embedding of spheroids

[0066] 1. Spheroids were immersed in formalin for 2 hours or overnight.

[0067] 2. They were then either embedded in agar and processed or placedin a piece of pre-folded tissue paper which was then folded and placedin a tissue-processing cassette. Alternatively a Cellsafe biopsycassette was used (a mesh chamber in which the spheroids are placed).The cassette was then closed and placed inside the processing cassette.The mesh prevented the spheroids escaping.

[0068] 3. The spheroid preparations were processed through ascendinggrades of alcohol to paraffin wax using a Citadel 2000 HistopathologyProcessing Unit.

[0069] 4. Sections of the paraffin wax blocks were cut using a microtomeonto coated sides.

[0070] D. Immunohistochemistry for CD68 (a pan macrophage marker) tolocalise macrophages in spheroids

[0071] 1. Spheroid sections were dewaxed in xylene and absolute alcohol.

[0072] 2. The endogenous peroxidase in the sections was then blockedwith 2% H202 in methanol for 10 mins.

[0073] 3. Antigen retrieval: sections were exposed to proteinases typeXXIV for 15 mins at 37° C.

[0074] The sections were then incubated in the following: (with 3×5 minwashes between each step).

[0075] 4. Normal serum for 30 mins at room temperature.

[0076] 5. Primary anti-CD68 monoclonal antisera at a dilution of 1:100for one hour at room temperature or overnight at 4° C.

[0077] 6. Secondary antibody (biotinylated horse anti-mouse IgG) for 30mins at room temperature followed by an avidin-biotin peroxidase complexfor 30 minutes at room temperature (ie using the Vector ABC Elite kit).

[0078] 7. Visualised with the chromagen. DAB or AEC for 20 10 mins.

[0079] 8. The nuclei were then counterstained with haematoxylin andsections mounted with coverslips for viewing.

[0080] NB: Spheroid sections needed to be washed thoroughly in diluentbetween each steep of the staining protocol to limitbackground/non-specific staining.

[0081] The imaging agent conjugate of choice can be infused (repeatedlyor as a single injection) into the general circulation so as to bind invivo to the surface of systemic mononuclear phagocytes and/ormacrophages already resident in diseased tissues (e.g. malignanttumours). Alternatively the imaging agent conjugate can be exposed tomonocytes ex vivo, following their purification from the blood ofpatients using such standard methods such as Ficoll-Hypaque gradientsand elutriation as described previously in (12). This method is asfollows:

[0082] 1. Ontain a fresh sample of venous blood in EDTA vacutainertubes. 15 ml will yield approximately 1 million monocytes.

[0083] 2. Dilute 1:1 with HBSS and overlay into an equal volume ofFicoll-Paque. (6×12 ml centrifuge tubes are recommended).

[0084] 3. Centrifuge at 600 g for 15 minutes.

[0085] 4. Remove plasma layer and carefully remove band of buffy coatcells. Resuspend cells in HBSS.

[0086] 5. To remove cell clumps pass cell suspension through 30 micronfilter.

[0087] 6. Wash cells by centrifuging at 300 g for 5 minutes andcompletely removing supernatant, then resuspend in 80 microlitres bufferper 10 million cells. Buffer=PBS supplemented with 2 mM EDTA and 0.5%BAS—degassed.

[0088] Homing of blood monocytes loaded up with imaging agent conjugatesinto malignant tumours can be augmented by prior treatment withconventional systemic therapies which induce local inflammation/necrosisin the diseased tissue (e.g. radiotherapy or chemotherapy in the case ofcancer patients). This stimulates the release of chemoattractant factorsfor monocytes/macrophages such as MCP-1 (13, 14) and would thus enhancethe delivery and hence the imaging of the conjugate at the diseasedsite.

[0089] Mode of production of selected imaging agent conjugates

EXAMPLE 1 Imaging Agent Conjugated to F(ab)₂ of a Monoclonal Antibody toCD87 (uPAR)

[0090] This conjugate uses a highly specific F(ab)₂ fragment, amonoclonal antibody to CD87 (urokinase plasminogen activator receptor;uPAR), to target naturally occurring uPAR on the surface of monocytesand macrophages.

[0091] A monoclonal antibody to CD87 is made as described in (15) andthen cleaved/purified to a specific F(ab)₂ monoclonal antibody fragmentusing standard proteolytic methods. Depending upon the part of uPAR usedto raise the antibody (i.e. as the antigen), the epitope for theantibody generated may either be in the (i) ligand (i.e. uPA)-bindingportion of the uPAR (in which case the imaging agent conjugate will onlybind to unoccupied uPAR on monocytes/macrophages), or (ii) the nonligand (i.e. uPA)-binding portion of the uPAR (in which case the imagingagent conjugate will bind to both occupied and unoccupied uPAR onmonocytes/macrophages). The most effective imaging agent uptake islikely to be achieved using the latter form of conjugate.

EXAMPLE 2 Imaging Agent Conjugated to PAI-2

[0092] This conjugate uses the affinity of plasminogen activatorinhibitor 2 (PAI-2) for urokinase plasminogen activator receptor(uPAR)—urokinase plasminogen activator complexes to target the imagingagent to the surface of monocytes and macrophages. PAI-2 triggers theinternalization of uPAR-uPA complexes, so the internalization by thesecells of the imaging agent attached to PAI-2 is assured.

[0093] Naturally occurring PAI-2 is obtained from the culturesupernatant of human blood monocytes stimulated maximally withinterleukin 1 or 2 as described in (16). This is then purified tohomogeneity in the usual manner by elution from an anti-PAI-2immunoaffinity column. Alternatively, PAI-2 can be produced in arecombinant expression system and purified according to the method of(17).

EXAMPLE 3 Imaging Agent Conjugated to CD14 Micro-Beads

[0094] The conjugate comprises an antibody that is specific for themacrophage surface molecule CD14. The antibody is conjugated to magneticmicrobeads and applied to monocytes as follows:

[0095] 1. Add 20 microlitres of CD14 microbeads per 10 million cells,mix and incubate for 15 minutes at 6-12 degrees C.

[0096] 2. Wash cells by adding 10-20×the labelling volume of buffer andcentrifuge at 300 g for 10 minutes, remove supernatant and resuspend in500 microlitres of buffer per 100 million cells.

[0097] 3. Choose correct column type (MS+ for 10 million total cells,VS+ for 100 million total cells for positive selection) and place in themagnet on the MiniMacs stand.

[0098] 4. Prepare column by flushing by 500 microlitres of buffer.

[0099] 5. Apply cell suspension and rinse with 3×500 microlitres ofbuffer.

[0100] 6. Remove column from separator, place column on a suitablecollection tube, pipette on 1 ml of buffer and flush out the positivecells using the plunger provided.

[0101] 7. Spheroids grown using the liquid overlay culture technique in96 well plates were allowed to reach maximal size of approximately 800microns.

[0102] 8. Remove approximately 100 microlitres of media from each wellof the plate and replace with the labelled monocyte suspension (50,000cells per ml to give 5,000 cells per spheroid).

[0103] 9. Allow monocytes to infiltrate overnight.

[0104] 10. Retrieve spheroids from 96 well plate and wash twice in PBSto remove loosely attached monocytes.

[0105] 11. Fix in 10% buffered formalin.

[0106] To detect the infiltrating monocytes the spheroids are analysedby magnetic resonance imaging (MRI).

[0107] These examples are represented as exemplary imaging agentconjugate candidates. It will be understood by those skilled in the artthat such conjugates represent selected examples and are not intended tolimit the scope of the invention.

[0108] The invention hereinbefore described therefore represents a mostelegant and effective means and method of delivering an imaging agent toa hypoxic or ischaemic site by use of monocytes and/or macrophages andtheir natural ability to congregate at a hypoxic or ischaemic site.

REFERENCES

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1. An imaging means comprising an imaging agent attached to an agentthat binds to a cell surface element of a mononuclear phagocyte.
 2. Animaging means according to claim 1 wherein said cell surface element isa protein, or part thereof.
 3. An imaging means according to claim 2wherein said protein is a receptor such as CD68, CD87, CSF-1, CD11B,CR3, MCP-1, 2 etch CD14, or mannose or mannose-6-phosphate receptor. 4.An imaging means according to any preceding claim wherein said bindingagent includes an antibody for binding to said cell surface element. 5.An imaging means according to claim 4 wherein said antibody ismonoclonal.
 6. An imaging means according to claims 4 or 5 wherein saidantibody is humanised.
 7. An imaging means according to any precedingclaim wherein said imaging agent comprises a hypoxia enhanced imagingagent.
 8. An imaging means according to any preceding claim wherein saidimaging agent is conjugated to a carrier molecule that promotesinternalisation of said agent into mononuclear phagocytes.
 9. An imagingmeans according to claim 8 wherein said carrier molecule includesplasminogen activator inhibitor (PAI-1 or PAI-2) or protease nexin (PN).10. A delivery system for targeting imaging compositions to hypoxic orischaemic sites comprising an imaging agent and, optionally, an agentfor controlling the functional effectiveness thereof, and coupledthereto, a binding agent for a cell surface element of a mononuclearphagocyte.
 11. A method for targeting imaging agents to hypoxic orischaemic sites comprising; i. coupling at least one of said agents to abinding agent of a cell surface molecule expressed by a mononuclearphagocyte; ii. exposing said coupled agents to mononuclear phagocytes;iii. allowing said phagocytes to migrate under conditions that supportmigration in vivo.
 12. A method for imaging hypoxic or ischaemic sitescomprising adminstering to the individual be treated the imaging meansaccording to claims 1-10.
 13. Mononuclear phagocytes having coupledthereto, or internalised therein, an imaging agent and an agent that isadapted to bind to a mononuclear phagocyte ligand which ligand istypically found on the cell surface of the said mononuclear phagocyte.